Cyanidin-3-glucoside binds to talin and modulates colon cancer cell adhesions and 3D growth.

Cyanidin-3-glucoside (C3G) is a natural pigment, found in many colorful fruits and vegetables. It has many health benefits, including anti-inflammation, cancer prevention, and anti-diabetes. Although C3G is assumed to be an antioxidant, it has been reported to affect cell-matrix adhesions. However, the underlying molecular mechanism is unknown. Here, we show that the expression of talin1, a key regulator of integrins and cell adhesions, negatively correlated with the survival rate of colon cancer patients and that depletion of talin1 inhibited 3D spheroid growth in colon cancer cells. Interestingly, C3G bound to talin and promoted the interaction of talin with ß1A-integrin. Molecular docking analysis shows that C3G binds to the interface of the talin-ß-integrin complex, acting as an allosteric regulator and altering the interaction between talin and integrin. Moreover, C3G promoted colon cancer cell attachment to fibronectin. While C3G had no significant effect on colon cancer cell proliferation, it significantly inhibited 3D spheroid growth in fibrin gel assays. Since C3G has no or very low toxicity, it could be potentially used for colon cancer prevention or therapy.

The NCI-N87 Cell Line as a Gastric Epithelial Model to Study Cellular Uptake, Trans-Epithelial Transport, and Gastric Anti-Inflammatory Properties of Anthocyanins.

Anthocyanins are ubiquitous plant pigments with reported antioxidant, anti-inflammatory, and anti-cancer activities. To better understand these benefits, metabolism of anthocyanins requires further evaluation, especially in the stomach. Mammalian cell cultures provide useful models for investigating compound metabolism and absorption, but they are generally maintained at physiological pH. The NCI-N87 cell line is an acid-stable model of the gastric epithelium used to study gastric drug metabolism. The objective of this work was to investigate the uptake, trans-epithelial transport, and anti-inflammatory activity of anthocyanins by the NCI-N87 cell line. The cells formed a coherent monolayer, stable ≤32 days post confluency. Minimal effects on monolayer integrity were observed when the pH of the apical chamber was adjusted to pH 3.0, 5.0, or 7.4. Anthocyanins were transported across the NCI-N87 cell monolayer at 37 °C, but not at 0 °C, suggesting a facilitated process. Chokeberry anthocyanins (0-1500 µM) were not cytotoxic. At apical pH 3.0, they had anti-inflammatory properties by significantly attenuating IL-8 secretion when added to medium before, during, and after incubation with IL-1ß. These results suggest that the NCI-N87 cell line is a physiologically relevant model for in vitro studies of the transport, anti-inflammatory and potential anti-carcinogenic activities of anthocyanins in gastric tissue.

The ameliorating effects of anthocyanins on the cross-linked signaling pathways of cancer dysregulated metabolism.

Cancer cells underlie the dysregulated metabolism of carbohydrate, lipid and protein and thereby, employ interconnected cross-linked signaling pathways to supply adequate energy for growth and related biosynthetic procedures. In the present study, a comprehensive review of cancer metabolism and anthocyanin's effect was conducted using the existing electronic databases, including Medline, PubMed, Scopus, and Web of Science, as well as related articles in the field. Such keywords as "cancer", and "cancer metabolism" in the title/abstract/keyword and all the "anthocyanins" in the whole text were used. Data were collected without time restriction until February 2020. The results indicated the involvement of several signaling pathways, including inflammatory PI3K/Akt/mTOR pathway, Bax/Bcl-2/caspases as apoptosis modulators, and NF-κB/Nrf2 as oxidative stress mediators in the cancer dysregulated metabolism. Compelling studies have shown that targeting these pathways, as critical hallmarks of cancer, plays a critical role in combating cancer dysregulated metabolism. The complexity of cancer metabolism signaling pathways, along with toxicity, high costs, and resistance to conventional drugs urge the need to investigate novel multi-target agents. Increasing evidence has introduced plant-derived secondary metabolites as hopeful anticancer candidates which target multiple dysregulated cross-linked pathways of cancer metabolism. Amongst these metabolites, anthocyanins have demonstrated positive anticancer effects by targeting inflammation, oxidative stress, and apoptotic signaling pathways. The current study revealed the cross-linked signaling pathways of cancer metabolism, as well as the promising pharmacological mechanisms of anthocyanins in targeting the aforementioned signaling mediators. To overcome the pharmacokinetic limitations of anthocyanins in cancer treatment, their interactions with gut microbiota and the need to develop related nano-formulations were also considered.

Polyphenols and bioavailability: an update.

Based on many cell culture, animal and human studies, it is well known that the most challenge issue for developing polyphenolics as chemoprevention or anti-diabtetic agents is the low oral bioavailability, which may be the major reason relating to its ambiguous therapeutic effects and large inter-individual variations in clinical trials. This review intends to highlight the unscientific evaluation on the basis of the published data regarding in vitro bioactivity of polyphenols, which may sometimes mislead the researchers and to conclude that: first, bio-accessibilities values obtained in the studies for polyphenols should be highly reconsidered in accordance with the abundant newly identified circulating and excreted metabolites, with a particular attention to colonic metabolic products which are obviously contributing much more than expected to their absorptions; second, it is phenolic metabolites, which are formed in the small intestine and hepatic cells,low molecular weight catabolic products of the colonic microflora to travel around the human body in the circulatory system or reach body tissues to elicit bioactive effects. It is concluded that better performed in vivo intervention and in vitro mechanistic studies are needed to fully understand how these molecules interact with human physiological and pathological processes.

Metabolism of anthocyanins and consequent effects on the gut microbiota.

Anthocyanins are natural water-soluble polyphenols present in fruits and vegetables. Health-promoting effects attributed to anthocyanins are mainly associated with oxidative stress inhibition and gut microbiota modulation. Dietary anthocyanins undergo a complex metabolism after ingestion and interact with endogenous and microbial enzymes, leading to the production of a large number of circulating and excreted anthocyanin metabolites and catabolic products. To date, the bioavailability and health benefits of anthocyanins have been widely documented. Although there are several papers that illustrated the metabolism of anthocyanins, the effects of dietary anthocyanins on the modulation of the gut microbial ecology and on the growth of certain microbial species are still poorly understood. The present paper summarizes the recent data on the absorption of anthocyanins in the upper gastrointestine and the metabolism of anthocyanins by gut microbiota. The modulatory effects of anthocyanins from different sources on gut microbiota are also discussed.

Biotransformation of anthocyanins from Vitis amurensis Rupr of "Beibinghong" extract by human intestinal microbiota.

1. Anthocyanins (ACNs) are a subclass of polyphenolic pigments belonging to the flavonoids and constitute an important group of human diets. There is accumulated evidence that consumption of ACN-rich diets such as Vitis amurensis Rupr of "Beibinghong" exerts protective activities against some human diseases. These beneficial properties may be due to the biotransformation by intestinal microbiota and its related bacterial dependent metabolism of ACNs. However, despite the compositional characterization of ACNs in extracts from V. amurensis Rupr, the biotransformation pathways of these compounds in the human intestinal tract have not been investigated so far. 2. In this study, the biotransformation of ACNs by the human intestinal microbiota and the derived metabolites were analyzed and characterized by RRLC-Q-TOF-MS and MS/MS methods. Eight kinds of ACNs were identified and could be bio-transformed under the action of human intestinal microbiota. The biotransformation pathway analysis showed that the microbiota acted by removing all glucosides to produce the corresponding aglycones, which were subsequently converted to phenolic acid and aldehydes. 3. These findings shed light on the mechanisms of ACNs degradation by the human intestinal microbiota and will lay a foundation for the industrial and pharmacological applications of the ACNs.

Particle size of milled chokeberry pomace did not influence in vitro cellular absorption and transport efficiencies of anthocyanins, phenolic acids and flavonols.

Industrial chokeberry pomace is very rich in polyphenols. The main focus here lies on the possible relationship between the particle size of chokeberry milled pomace and an enhanced absorption and transport of polyphenols by Caco-2 cells. Wet milling was used to produce materials with particle size distributions in the micrometre and in the sub-micrometre to nanometre ranges starting from chokeberry pomace. Milled materials with about 50% of the particles with a mean size (x50,3) of 223 ± 13 µm (coarse milling) and about 90% of the particles with x50,3 of 160 ± 40 nm (fine milling, sonication) were obtained. None of the milled materials exhibited cytotoxic effects within the tested concentration-ranges. The polyphenol absorption and the transport efficiencies from the fine and the coarse milled materials were similar. Thus, no effect of the particle size upon cellular uptake and transport could be established, but agglomeration of particle during incubation cannot be excluded as the cause. Furthermore, based on polyphenol stability we postulate that direct milling may be applied to valorise the processing by-product from commercial fruit juice production.

Anthocyanins and Their C6-C3-C6 Metabolites in Humans and Animals.

Research on the bioavailability of anthocyanins has focused, historically, on the non-flavonoid (C6-Cn) products that arise from anthocyanins in vivo. However, this review focuses on the products of anthocyanins that still possess the flavonoid structure (C6-C3-C6). Described herein are aspects of the in vivo pool of C6-C3-C6 anthocyanin-derived intermediates. Properties related to molecular size, shape, and polarity conveyed by six major anthocyanidin structures are discussed. The presence of a glycoside or not, and a variety of possible phase 2 conjugates, gives rise to a chemically diverse pool of C6-C3-C6 intermediates. Chemical properties influence the in vivo stability of anthocyanin-derived products, as well as their suitability as a substrate for xenobiotic conjugation and transport, and their association with the biomatrix. The flavonoid structure is associated with bioactivity and the particular properties of these C6-C3-C6 products of anthocyanins determines their deposition in the body, which may influence in vivo processes and ultimately health outcomes.

Contribution of Berry Polyphenols to the Human Metabolome.

Diets rich in berries provide health benefits, however, the contribution of berry phytochemicals to the human metabolome is largely unknown. The present study aimed to establish the impact of berry phytochemicals on the human metabolome. A "systematic review strategy" was utilized to characterize the phytochemical composition of the berries most commonly consumed in the USA; (poly)phenols, primarily anthocyanins, comprised the majority of reported plant secondary metabolites. A reference standard library and tandem mass spectrometry (MS/MS) quantitative metabolomics methodology were developed and applied to serum/plasma samples from a blueberry and a strawberry intervention, revealing a diversity of benzoic, cinnamic, phenylacetic, 3-(phenyl)propanoic and hippuric acids, and benzyldehydes. 3-Phenylpropanoic, 2-hydroxybenzoic, and hippuric acid were highly abundant (mean > 1 µM). Few metabolites at concentrations above 100 nM changed significantly in either intervention. Significant intervention effects (P < 0.05) were observed for plasma/serum 2-hydroxybenzoic acid and hippuric acid in the blueberry intervention, and for 3-methoxyphenylacetic acid and 4-hydroxyphenylacetic acid in the strawberry intervention. However, significant within-group effects for change from baseline were prevalent, suggesting that high inter-individual variability precluded significant treatment effects. Berry consumption in general appears to cause a fluctuation in the pools of small molecule metabolites already present at baseline, rather than the appearance of unique berry-derived metabolites, which likely reflects the ubiquitous nature of (poly)phenols in the background diet.

An LC-MS/MS method for quantitation of cyanidin-3-O-glucoside in rat plasma: Application to a comparative pharmacokinetic study in normal and streptozotocin-induced diabetic rats.

A sensitive and reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to determine cyanidin-3-O-glucoside (Cy-3G) in normal and streptozotocin-induced diabetic rat plasma. Chromatographic separation was carried out on a Zorbax SB-C18 (50 × 4.6 mm, 5 µm) column and mass spectrometric analysis was performed using a Thermo Finnigan TSQ Quantum Ultra triple-quadrupole mass spectrometer coupled with an ESI source in the negative ion mode. Selected reaction monitoring mode was applied for quantification using target fragment ions m/z 447.3 → 285.2 for Cy-3G and m/z 463.0 → 300.1 for quercetin-3-O-glucoside (internal standard). The calibration curve was linear over the range 3.00-2700 ng/mL (r2 ≥ 0.99) with the lower limit of quantitation at 3.00 ng/mL. Intra- and inter-day precision was <14.5% and mean accuracy was from -11.5 to 13.6%. Stability testing showed that Cy-3G remained stable during the whole analytical procedure. After validation, the assay was successfully used to support a preclinical pharmacokinetic comparison of Cy-3G between normal and diabetic rats. Results indicated that diabetes mellitus significantly altered the in vivo pharmacokinetic characteristics of Cy-3G after oral administration in rats.

The Chemical Reactivity of Anthocyanins and Its Consequences in Food Science and Nutrition.

Owing to their specific pyrylium nucleus (C-ring), anthocyanins express a much richer chemical reactivity than the other flavonoid classes. For instance, anthocyanins are weak diacids, hard and soft electrophiles, nucleophiles, prone to developing π-stacking interactions, and bind hard metal ions. They also display the usual chemical properties of polyphenols, such as electron donation and affinity for proteins. In this review, these properties are revisited through a variety of examples and discussed in relation to their consequences in food and in nutrition with an emphasis on the transformations occurring upon storage or thermal treatment and on the catabolism of anthocyanins in humans, which is of critical importance for interpreting their effects on health.

The Microencapsulation of Maqui (Aristotelia chilensis (Mol.) Stuntz) Juice by Spray-Drying and Freeze-Drying Produces Powders with Similar Anthocyanin Stability and Bioaccessibility.

The microencapsulation of maqui juice by spray-drying and freeze-drying was studied as a strategy to protect anthocyanins in new food formulations in order to improve the anthocyanin retention before consumption and the bioaccessibility. It is well known that the encapsulation method affects both the shape and size of powders, being assumed that undefined forms of freeze-drying powders might affect their stability due to the high permeability to oxygen. The objective of this study was to compare the microencapsulation of maqui juice by spray-drying and freeze-drying, evaluating the stability of specific anthocyanins in yogurt and after in vitro digestion. Results indicated that most relevant differences between spray-drying and freeze-drying powders were the morphology and particle size that affect their solubility (70.4⁻59.5%) when they were reconstituted in water. Nevertheless these differences did not affect the stability of anthocyanins as other research have proposed. Both encapsulation methods generated powders with a high stability of 3-O-monoglycosylated anthocyanins in yogurt (half-life values of 75⁻69 days for delphinidin-3-sambubioside). Furthermore, no significant differences in the bioaccessibility of anthocyanins between maqui juice powders (44.1⁻43.8%) were found. In conclusion, the microencapsulation of maqui juice by freeze-drying is as effective as spray-drying to produce new value-added food formulations with stable anthocyanins.

Effect of In Vitro Digestion on Water-in-Oil-in-Water Emulsions Containing Anthocyanins from Grape Skin Powder.

The effects of in vitro batch digestion on water-in-oil-in-water (W/O/W) double emulsions encapsulated with anthocyanins (ACNs) from grape skin were investigated. The double emulsions exhibited the monomodal distribution (d = 686 ± 25 nm) showing relatively high encapsulation efficiency (87.74 ± 3.12%). After in vitro mouth digestion, the droplet size (d = 771 ± 26 nm) was significantly increased (p < 0.05). The double W1/O/W2 emulsions became a single W1/O emulsion due to proteolysis, which were coalesced together to form big particles with significant increases (p < 0.01) of average droplet sizes (d > 5 µm) after gastric digestion. During intestinal digestion, W1/O droplets were broken to give empty oil droplets and released ACNs in inner water phase, and the average droplet sizes (d < 260 nm) decreased significantly (p < 0.05). Our results indicated that ACNs were effectively protected by W/O/W double emulsions against in vitro mouth digestion and gastric, and were delivered in the simulated small intestine phase.

A theoretical physiologically based pharmacokinetic approach for modeling the fate of anthocyanins in vivo.

Recent studies on the pharmacokinetics of anthocyanins (ACNs) and their metabolites have uncovered evidence for hitherto unknown physiological effects affecting the fate of these compounds in vivo. In particular, it has been shown that the stomach, in addition to the small intestine, has an important role in absorption. Most studies still use a noncompartmental or one-compartmental approach to determine the pharmacokinetic parameters of ACNs, which does not represent the anatomical and physiological conditions that a compound is subject to in the organism. Thus, the objective of this study was to review the current knowledge of the different processes involved in the metabolism of ACNs once ingested and, based on this information, propose a theoretical physiologically based, multicompartmental pharmacokinetic (PBMK) model to describe their fate in vivo. This is the first study that reports a PBMK model for ACNs; the model provides a more physiologically representative approach for ANC metabolism, which could be used as a basis for experimental designs and interspecies scale-up.

Time, Concentration, and pH-Dependent Transport and Uptake of Anthocyanins in a Human Gastric Epithelial (NCI-N87) Cell Line.

Anthocyanins are the largest class of water soluble plant pigments and a common part of the human diet. They may have many potential health benefits, including antioxidant, anti-inflammatory, anti-cancer, and cardioprotective activities. However, anthocyanin metabolism is not well understood. Studies suggest that anthocyanins absorption may occur in the stomach, in which the acidic pH favors anthocyanin stability. A gastric epithelial cell line (NCI-N87) has been used to study the behavior of anthocyanins at a pH range of 3.0-7.4. This work examines the effects of time (0-3 h), concentration (50-1500 µM), and pH (3.0, 5.0, 7.4) on the transport and uptake of anthocyanins using NCI-N87 cells. Anthocyanins were transported from the apical to basolateral side of NCI-N87 cells in time and dose dependent manners. Over the treatment time of 3 h the rate of transport increased, especially with higher anthocyanin concentrations. The non-linear rate of transport may suggest an active mechanism for the transport of anthocyanins across the NCI-N87 monolayer. At apical pH 3.0, higher anthocyanin transport was observed compared to pH 5.0 and 7.4. Reduced transport of anthocyanins was found to occur at apical pH 5.0.

The encapsulation of anthocyanins from berry-type fruits. Trends in foods.

During the last decade, many berry-type fruits have been recognised as good sources of anthocyanins. Nevertheless, the use of anthocyanins in the development of food colourants and healthy and/or functional ingredients has been limited because of their low stability under given environmental conditions and interaction with other compounds in the food matrix. This review compiles information about the encapsulation of anthocyanins from twelve different berry-type fruit species as a technology for improving the stability and/or bioavailability of anthocyanins. Encapsulation by spray drying has been the primary method used to encapsulate anthocyanins, and some studies attempt to keep anthocyanin microparticles stable during storage. Nevertheless, more studies are needed to determine the stability of anthocyanin microparticles in food matrices over the product shelf life in the development of food colourants. Studies about encapsulated anthocyanins in simulated gastrointestinal models have primarily been conducted on the release of anthocyanins from microparticles to evaluate their bioavailability. However, adding anthocyanin microparticles to a food vehicle must guarantee the health properties attributed to the specific anthocyanins present in berry-type fruits.

Bioavailability of anthocyanins.

Anthocyanins are a subgroup of flavonoids responsible for the blue, purple, and red color of many fruits, flowers, and leaves. Consumption of foods rich in anthocyanins has been associated with a reduced risk of cardiovascular disease and cancer. The fate of anthocyanins after oral administration follows a unique pattern rather different from those of other flavonoids. Anthocyanins could be absorbed from the stomach as well as intestines. Active transporters may play a role in the absorption of anthocyanins from the stomach as well as in their transfer within the kidney or liver. Anthocyanins such as cyanidin-3-glucoside and pelargonidin-3-glucoside could be absorbed in their intact form into the gastrointestinal wall; undergo extensive first-pass metabolism; and enter the systemic circulation as metabolites. Phenolic acid metabolites were found in the blood stream in much higher concentrations than their parent compounds. These metabolites could be responsible for the health benefits associated with anthocyanins. Some anthocyanins can reach the large intestine in significant amounts and undergo decomposition catalyzed by microbiota. In turn, these decomposition products may contribute to the health effects associated with anthocyanins in the large intestine. This review comprehensively summarizes the existing knowledge about absorption, distribution, metabolism, and elimination of anthocyanins as well as their decomposition within the gastrointestinal lumen.

Identification of brain-targeted bioactive dietary quercetin-3-O-glucuronide as a novel intervention for Alzheimer's disease.

Epidemiological and preclinical studies indicate that polyphenol intake from moderate consumption of red wines may lower the relative risk for developing Alzheimer's disease (AD) dementia. There is limited information regarding the specific biological activities and cellular and molecular mechanisms by which wine polyphenolic components might modulate AD. We assessed accumulations of polyphenols in the rat brain following oral dosage with a Cabernet Sauvignon red wine and tested brain-targeted polyphenols for potential beneficial AD disease-modifying activities. We identified accumulations of select polyphenolic metabolites in the brain. We demonstrated that, in comparison to vehicle-control treatment, one of the brain-targeted polyphenol metabolites, quercetin-3-O-glucuronide, significantly reduced the generation of ß-amyloid (Aß) peptides by primary neuron cultures generated from the Tg2576 AD mouse model. Another brain-targeted metabolite, malvidin-3-O-glucoside, had no detectable effect on Aß generation. Moreover, in an in vitro analysis using the photo-induced cross-linking of unmodified proteins (PICUP) technique, we found that quercetin-3-O-glucuronide is also capable of interfering with the initial protein-protein interaction of Aß(1-40) and Aß(1-42) that is necessary for the formation of neurotoxic oligomeric Aß species. Lastly, we found that quercetin-3-O-glucuronide treatment, compared to vehicle-control treatment, significantly improved AD-type deficits in hippocampal formation basal synaptic transmission and long-term potentiation, possibly through mechanisms involving the activation of the c-Jun N-terminal kinases and the mitogen-activated protein kinase signaling pathways. Brain-targeted quercetin-3-O-glucuronide may simultaneously modulate multiple independent AD disease-modifying mechanisms and, as such, may contribute to the benefits of dietary supplementation with red wines as an effective intervention for AD.

Structure-activity relationships of anthocyanidin glycosylation.

This paper summarizes the main achievements about the structure-activity relationships of anthocyanidin glycosylation. Anthocyanidin glycosylation is the essential step of anthocyanin biosynthesis and also the prerequisite of the further modifications of anthocyanins, which is jointly characterized by the glycosylation site, the type and number of the glycosyl as well as the glycosidic bond type. It generally enhances the stability, results in the hypsochromic effect and blueing, decreases the bioavailability and anticancer activity, and decreases, increases, or does not change the antioxidant activity of the anthocyanidins or anthocyanins, which is synergetically determined by the glycosylation site and the type and number of the glycosyl. Thereinto, in nature, the blue hues caused by the glycosylation may also be reinforced by the formation of the anthocyanic vacuolar inclusions. This review could provide a reference for the research of the structure-optimizing and function-exploiting of anthocyanins.

Practical application of flavonoid-poor menu meals to the study of the bioavailability of bilberry anthocyanins in human subjects.

Practical application of flavonoid-poor menus was evaluated on the bioavailability of anthocyanins as model flavonoids. Detectable amounts of flavonoids were not found in plasma and urine collected from 13 participants, who took the menus. After ingesting bilberry anthocyanins (919 µmol), average plasma AUC0-6h, Cmax, Tmax values and urinary recovery were 386.0 nmol h/mL, 139.1 nM, 1.31 h and 0.21%, respectively.